Tacrolimus (FK506), an inhibitor of calcineurin, is an immunosuppressive agent used in clinical trials of transplant patients. Although FK506 targets Ca(2+)-mediated T-cell signaling, phenotype(s) of the specific target cells and the corresponding cytokine pathways are not well known. In this study, the impact of FK506 on number and characteristic of T-cells in selected lymphoid tissues of gnotobiotic (GB) piglets was determined. FK506-treated GB piglets were compared with untreated GB and conventional piglets. The T-helper, cytotoxic, natural killer, double-positive, and activated T-cell populations were analyzed in suspensions of mononuclear cells isolated from thymus, mesenteric lymph nodes and peripheral blood. In vitro secretion of interleukin-8 and interferon-gamma in concanavalin A-stimulated lymphoid cell-cultures was measured by ELISA. Daily intramuscular treatment of GB piglets with 1mg/kg of FK506 from birth for 4 weeks resulted in lowered (P<0.05) in vitro secretion of interferon-gamma and interleukin-8. Moreover, depletions of MNC in systemic and mucosa-associated lymphoid tissues were observed in piglets treated with FK506. The depletions of mononuclear cells and low levels of interferon-gamma and interleukin-8 in piglets treated with FK506 were accompanied by lower proportion of CD3+, CD2+CD4+ and CD2+CD8+ T-cell phenotypes in peripheral blood but not in thymus and mesenteric lymph nodes. These results indicate that FK506-treatment causes immunosuppression in GB piglet, and this effect could be exploited further to study opportunistic pathogens in pig model.